ES2235958T3 - NUCLEIC ACID AMPLIFICATION REACTION STATION FOR DISPOSABLE TEST DEVICES. - Google Patents

Abstract

An instrument for conducting nucleic acid amplification reactions in a disposable test device. The test device includes a first reaction chamber containing a first nucleic acid amplification reagent (e.g., primers and nucleotides) and a second reaction chamber either containing, or in fluid communication, with a second nucleic acid amplification reagent (e.g., an amplification enzyme such as RT). The instrument includes a support structure receiving the test device. A temperature control system maintains the first reaction chamber at a first elevated temperature but simultaneously maintains the second nucleic acid amplification reagent at a second temperature lower than the first temperature so as to preserve the second nucleic acid amplification reagent. An actuator operates on a fluid conduit in the test device to place the first and second reaction chambers in fluid communication with each other after a reaction has occurred in the first reaction chamber at the first temperature. A pneumatic system is also provided that assists in fluid transfer of a reaction solution from the first chamber to the second chamber.

Description

Acid Amplification Reaction Station nucleic for disposable test devices.

This invention relates to the field of procedures and devices for carrying out reactions of nucleic acid amplification, and more particularly, this invention refers to an automated instrument to carry conduct nucleic acid amplification reactions.

Description of the related technique

Today based amplification reactions in nucleic acid they are widely used in clinical laboratories and research for the detection of genetic diseases and infectious The amplification schemes that are currently know can be classified into two categories, based on whether, after an initial stage of denaturation (which is typical is perform at a temperature of ≥ 65 degrees C) for DNA amplifications or for RNA amplifications, which involve a large amount of secondary initial structure, the reactions are conducted by means of a continuous cycle of the temperature between the denaturation temperature and a pinching and synthesis annealing temperature (or activity amplicon polymerase) ("cyclic reactions"), or if the temperature remains constant throughout the entire Enzymatic amplification procedure ("reactions isothermal "). Typical cyclic reactions are the Reaction in Polymerase and Ligase chain (PCR and CSF, respectively), Representative schemes of isothermal reactions are the NASBA (amplification based on the nucleic acid sequence), the transcription amplification (TMA) and braided displacement amplification (SDA). The reaction occurs, in isothermal reactions after the initial stage of denaturation (if required), at a constant temperature, being typical at a lower temperature at which the reaction of the Enzymatic amplification is optimized.

Before the discovery of thermostable enzymes, the methodologies that used the temperature cycle were seriously hindered by the need to dispense free polymerase into a tube for amplification (such as a test tube) after each cycle of denaturation, since the high temperature that polymerase was inactivated for denatured during each cycle. With the discovery of thermostable Taq polymerase ( Thermophilus aquaticus ) a considerable simplification of the PCR analysis procedure was achieved. This improvement eliminated the need to open the amplification tubes after each amplification cycle to add pure enzyme. This led to the reduction of both the risk of contamination and the costs related to enzymes. The introduction of thermostable enzymes has also allowed the relatively simple automation of the PCR technique. In addition, this new enzyme allowed the implantation of simple disposable devices (such as a single tube) for use with temperature cycling equipment.

The TMA requires the combined activities of, at less, two (2) enzymes for which they have not yet been discovered optimal thermostable variants. For optimal annealing presser in the reaction of the TMA an initial stage of denaturation (at a temperature of ≥ 65 degrees C) for eliminate the second structure of the objective. Then the reaction mixture to a temperature of 42 degrees C to allow pinching annealing. This temperature is also the optimal reaction temperature for the combined activities of T7 polymerase of RNA and reverse transcriptase (RT), which include an endogenous activity of RNase H or, alternatively, It is provided by another reagent. The temperature is maintained at 42 degrees C throughout the following isothermal amplification reaction. However, the denaturation stage, which precedes the cycle of amplification forces the user to add the enzyme to the test tube after the cooling period in order to avoid the inactivation of enzymes, therefore it is necessary that the stage of the denaturation is carried out separately from the stage of amplification

According to current practice the test tube, after add the sample for test or control, or both, to the mixture of amplification reagent (which is typically containing the nucleotides and scavengers), is subjected to temperatures of ≥ 65 degrees C and then cooled to the amplification temperature of 42 degrees C. Then the enzyme is added by hand to start the amplification reaction. It is typical that this stage requires the opening of the amplification tube. Not only the opening of the tube of amplification, to add the enzyme, or subsequent adding an enzyme to an open tube is inconvenient, but which also increases the risk of contamination.

An alternative solution for amplification of A DNA sample is described in US Pat. 5,270,183 from Corbett et al. In this technique, the reaction mixture is injected into a stream of carrier fluid, then the fluid carrier passes through a plurality of temperature zones in the which polymerase chain reactions take place. The temperature of the different zones and the time that elapses for that the carrier fluid crosses the temperature zones is controlled in such a way that the three events occur: the denaturation of DNA strands, annealing of oligonucleotine trappers to complementary sequences in DNA and the synthesis of new strands of DNA. To carry out the Patent procedure 5,270,183 a tube is provided, the associated temperature zones and pumping means.

In U.S. Pat. US-A-5,270,183 discloses a apparatus for carrying out the procedure, said said comprising apparatus a heating system to provide different temperatures in the reaction chambers, a means to control the passage of fluid between the chambers and a vacuum subsystem to extract fluid between the chambers by means of vacuum probes in tip.

The present invention provides a system of nucleic acid amplification reaction that substantially eliminates the risk of contamination and provides a solution comfortable, simple and easy to use for the reactions of the nucleic acid amplification. With the analysis devices and the amplifying station according to the present invention achieves the integration of the denaturation stage with the stage of the amplification without the need for manual transfer of the enzyme and without Exposure of the amplification chamber to the environment. The contamination risks from sample contamination The sample inside the processing station is avoided since the amplification reaction chamber is airtight and does not open to introduce the patient sample to the enzyme. The pollution from environmental sources is avoided since The amplification reaction chamber remains airtight. He risk of contamination in acid amplification reactions Nucleide is, especially, critical as products of amplification in large quantities.

Summary of the invention

In a first aspect, a station is provided for perform a nucleic acid amplification reaction that carried out in a unit and disposable analysis device. He analyzer device has a first reaction chamber that contains a first nucleic acid amplification reagent (such as pinchers and nucleotides) and a second chamber of reaction that contains, or is in fluid communication with, a second nucleic acid amplification reagent (for example, a amplifying enzyme such as RT).

The station includes a support structure that Receive the analyzer device. Support structure comprises, in the illustrated embodiment, a set of easels erect receiving a test strip, disposable, containing The reaction chambers. The station also includes a system for controlling the temperature of the analyzer device. This temperature control system maintains the first chamber of the reaction at a high first temperature, in which the reaction takes place in the first reaction chamber between a first sample or objective and the first amplification reagent. Without However, the temperature control system maintains simultaneously the second reagent of the amplification reaction of the nucleic acid at a second temperature lower than said first temperature in order to preserve said second reagent of the nucleic acid amplification reaction. In the realization illustrated the temperature control system comprises a pair of thermoelectric elements coupled to the structure of support.

The station also comprises an actuator operating on the analyzer device to place the first and the second reaction chambers in fluid communication with each other. Normally the first and second reaction chambers are isolated from each other by means of a closed valve inside a connecting duct that links the first and second cameras The actuator is operational in the analyzer device after the reaction has occurred in the first chamber to the First temperature A second reaction portion of nucleic acid amplification, for example, amplification of target sequences of RNA or DNA, occurs in the second chamber with the second reagent of the nucleic acid amplification reaction. The second nucleic acid amplification reagent is preserves thanks to the maintenance of this reagent to the second temperature (i.e. lower) while the reaction in the first chamber takes effect at the first temperature (i.e. highest).

As described here, the station amplification can be designed to simultaneously process a multitude of analyzer devices. The support structure, the temperature control system and actuators are designed, in this embodiment, to operate simultaneously in All analyzer devices.

After the reaction between the fluid sample and the reagents in the first reaction chamber, the solution of The reaction is conducted within the second reaction chamber. According the present invention, the support structure works with a vacuum housing that is lowered over the support structure to form a vacuum enclosure around the devices analyzers A vacuum is removed from the vacuum enclosure. When he vacuum is released, a pressure gradient between the first and the second reaction chambers makes the reaction dissolution flow between the first and second chambers of the reaction.

The apparatus according to this invention is defined in the claim 1.

Thus, in a second aspect of the invention, provides an amplifying station to carry out a plurality of nucleic acid amplification reactions in a plurality of disposable analyzer devices. This station amplifier comprises a support structure adapted to receive a plurality of analyzer devices and a system of temperature control for analyzer devices, a actuator set and a compressed air system. System temperature control maintains the temperature of the analyzer devices according to the profile (or profiles) that are desired (or desired) for the acid amplification reaction nucleic. The actuator assembly works in each of the analyzer devices for opening a fluid conduit in the analyzer devices and thus allow a reaction solution flow from a first location in the analyzer device (for example, a first chamber of the reaction) to a second location in said device analyzer (for example, a second reaction chamber that contain an amplifying enzyme). Compressed air system works on analyzer devices to extract a dissolution of the reaction from the first location to the second location, after the actuator assembly has operated in the analyzer devices to put the first and second portions in fluid communication with each other.

The amplification station includes, in a possible embodiment of this invention, a stirring system mechanic that agitates the analyzer devices to thereby encourage mixing of the reaction solution and reagents in the first and second chambers of the reaction.

The form factor of the analyzer device in the Amplification station is not considered to be critical. In the illustrated embodiment the analyzer device takes the form of a test strip that is compatible with a detection instrument analytical fluid transfer, currently available, to know the VIDAS® instrument, manufactured and distributed by the Assignee of the present invention, bioMérieux, Inc. Thus, provide the analyzer devices in a size and shape factor that use easily on an existing instrument base or selected allows instruments to be marketed and used very much with a reduction in capital investment, and without having to develop another instrument to process the reaction and detect the resulting amplifiers. However, it will be clear that, Starting from the detailed description that follows, this invention is you can practice with other configurations and form factors other than the preferred embodiment now described with detail here.

These and many other aspects and configurations of this invention will be readily understood by the following detailed description.

Brief description of the drawings

An embodiment is described below. presently preferred of this invention together with the figures of the attached drawings, in which the reference numbers correspond to similar elements in the various views, in which:

Figure 1 is a perspective view of a amplification reaction station according to one embodiment preferred of this invention;

Figure 1A is a perspective view of a test strip and the associated cover member that is used with the amplification reaction station of this invention in the Figure 1;

Figure 2 is another perspective view of the test strip and the cover member of Figure 1A, which shows the cover member attached to the test strip and with a portion of the erect or raised lid member, allowing access to the first reaction chamber of the two reaction vessel cameras inside it;

Figure 3 is another perspective view of the analysis strip of figure 2;

Figure 4 is an isolated, perspective view, of the cover member of Figures 2 and 3, shown from down;

Figure 4A is a perspective view of a alternative embodiment of the cover member of Figure 4 showing a hand operated button provided to pierce the thin membrane of the cover covering the chamber A of the analysis strip of the figure 1A;

Figure 4B is an isolated perspective view. of the cover member of Figure 4A shown from below, illustrating an outstanding point that pierces the membrane when press the button in figure 4A;

Figure 5 is a view from the top of the strip of analysis of figures 2 and 3;

Figure 6 is a sectional view of the strip of analysis of figure 5, shown along the line 6-6 of Figure 5;

Figure 7 is a sectional view of the strip of analysis of figure 5, shown along the line 7-7 of Figure 5;

Figure 8 is a plan view of the strip of analysis of figure 5;

Figure 9 is a detailed elevation view of the upper portion of the test strip in the region adjacent to the second reaction vessel, showing the configuration in the side of the strip that is securely held by the legs elastic of the cover member to close the cover member in the analysis strip;

Figure 10 is a detailed sectional view of the test strip, partly detached, illustrating the closure configuration shown in figure 9;

Figure 11 is a detailed plan view of the test strip of figure 5 in the duct region of connection that links the first reaction chamber with the second reaction chamber;

Figure 12 is a sectional view of a portion of the analysis strip of figures 5 and 11 taken along the line 12-12 of figure 13, that is, along of the long axis of the test strip in the region of the duct connection that links the first reaction chamber to the second reaction chamber, showing the placement of a ball inside of the connecting duct that acts just like a shut-off valve the connection conduit;

Figure 13 is a sectional view of a portion of the test strip of figure 5 taken in an orthogonal direction to the long axis of the capsule, along the line 13-13 of Figures 11 and 12;

Figure 14 is a perspective view of the analysis strip of the class shown in figure 5 with a fork gadget used to open the connection conduit, with the arrow indicating the relative movement of the fork with regarding the test strip and the dashed lines indicating the insertion of the fork teeth into the strip of analysis to open the ball valve in the duct Connection;

Figure 15 is a schematic illustration of a vacuum station that incorporates heat sinks for the analysis strip and with a housing that meshes a structure of support to form a vacuum enclosure around the strips of analysis, with each strip associated with a fork to open the connection duct when the vacuum chamber housing lower and engage the support structure;

Figure 16 is a sectional view of the strip of analysis of figure 5 taken in a transverse direction to the axis along the test strip in the vicinity of the conduit of connection, showing the action of the forks of figures 14 and 15 deforming the material of the connecting conduit for, of this mode, open the valve;

Figure 17 is a sectional view of the strip of analysis of figure 16 showing the deformation of the conduit of connection and the passage of fluid through the connection conduit;

Figure 18 is a perspective view of the instrument of figure 1 with the top and side panels removed in order to show the details of the two cameras and of the compressed air system;

Figure 19 is a perspective view of the instrument of figures 1 and 18 as seen from behind;

Figure 20 is a perspective view of a of the stations in the instrument of Figure 1A, shown isolated from the rest of the instrument to better illustrate the mechanical configuration thereof;

Figure 21 is an elevation view of the station of figure 20 shown from the rear side of the same;

Figure 22 is a side elevational view of the station of figure 20;

Figure 23 is another perspective view of the station of figure 20;

Figure 24 is another perspective view of the station of figure 20 shown from below and in front of the station, showing the belt drive mechanism that controls the rise and fall of the vacuum enclosure housing and the mechanical agitation of the test strips;

Figure 25 is a side elevational view of the station of figure 20, shown from the opposite side of the figure 22;

Figure 26 is a front elevational view of the station of figure 20;

Figure 27 is a plan view from above of the station of figure 20;

Figure 28 is a vertical sectional view of the station of figure 20, taken along the line 28-28 of Figures 25 and 27;

Figures 29A and 29B are perspective views. of the vacuum housing of figures 10 to 28 which descends on top of the support structure that carries the test strips in order to form a vacuum enclosure around the strips of analysis;

Figure 29C is a sectional view of the vacuum housing of figure 29A;

Figures 30A to 30D are various views of the actuator assembly of figure 28 that actuates the valves within the analysis strips to allow a dissolution of the reaction flow from the first chamber of the reaction vessel of two cameras, arranged inside it, until the second camera;

Figure 31 is a side view, partly in section, of a portion of the vacuum housing of figures 29A and 29B in an upright position in relation to the support structure which holds the test strips;

Figures 32A to 32D are various views of the arrangement of an optical sensor positioned above the support structure in order to detect if the user has installed a test strip in each of the slots of the support structure;

Figures 33A, 33B and 33C are several views of the support structure of figure 20 that holds the strips of analysis within the station;

Figure 34 is a bottom plan view above the support structure of Figure 33A showing the position of the thermoelectric elements and the heatsinks of heat for the test strips, which keep the container two chambers at the appropriate temperatures;

Figure 35 is a schematic illustration of the operation of the thermoelectric elements of the figure 3. 4;

Figure 36 is a sectional view of the member of tray holder of figure 33A taken along the line 36-36;

Figure 37 is a sectional view of the member of tray holder of figure 33A taken along the line 37-37 of figure 34 showing the elements thermoelectric and heat sinks;

Figure 38 is a more detailed sectional view. of the support structure on the right side of Figure 37;

Figure 39 is another sectional view of the support structure of Figure 33C, taken along the line 39-39 of figures 33C and 34;

Figure 40 is a perspective view of the superstructure of the station with most of the pieces of the same disassembled in order to better illustrate the systems of station transmission;

Figure 41A is a perspective view, insulated, of the horizontal support member and the regulating screw of figure 28; Figure 41B is a sectional view of the member of bracket and collar of figure 41A;

Figure 42 is a perspective view of the transmission screws of figure 40, shown from below;

Figure 43 is a bottom plan view above the transmission systems shown in figures 40 and 42;

Figure 44 is a sectional view of the system transmission of figure 40;

Figure 45 is a schematic principle of electrical system for the station of figure 20;

Figure 46 is a schematic principle of compressed air system for the station of figure 20;

Figure 47 is a schematic and a graph showing a thermal cycle representative of the station of figure 20.

Detailed description of the preferred embodiment I. General Compendium

With reference now to figure 1, a preferred embodiment of the instrument to control the reactions of nucleic acid amplification in an analyzer device disposable is generally indicated by the reference number 1. Figures 1 to 17 show an analyzer device disposable and described here extensively. One or more disposable analyzer devices, such as one to six or of six to twelve in figure 2, are introduced by hand into instrument 1 and they are installed in the support structures within it. The disposable analyzer devices contain cameras of the amplification reaction, reagents and a sample for the reaction of nucleic acid amplification.

Instrument 1 includes an amplifier module 2 with two boxes 3, designated box A and box B. More can be added modules containing more boxes as desired to increase the Sample production Each box 3 acts as an opening for a amplifier station 200 located inside the module amplifier 2. Amplifier stations 200 are shown with more details in figures 20 and following. The amplifier module 2 contains the mechanical, compressed air, temperature systems and electrical that control an acid amplification reaction nucleic that occurs in the analyzer device of figure 2. These systems will be described with extension below.

The amplifier module 2 is linked by from an RS-232 cable 4 to a computer system 5, for general uses, which includes a central processing unit 6 and a user interface 7. The CPU (central processing unit) is loaded with a software program that allows a technician control the operation of station 1 via the interface of the user 7. The CPU is, in a preferred embodiment, incorporated inside module 2. Computer system 5 can be linked more of an amplifier module 2 in one more capacity implementation high. An additional amplifier module with three boxes (resulting in a total of 5 boxes) could be linked to the computer system. The Menu screens in user interface 7 allow the operator check the operation of each box inside module 2 or in a extension module that can be added. The system described is versatile and can be adapted to the user's requirements to Various analysis situations.

After the amplification reaction of nucleic acid has been performed on the analyzer devices disposable, introduced in boxes 3 of instrument 1, the devices are removed by hand from instrument 1 and moved to another instrument to hybridize amplification products to one or more probes, for example, a detection probe and a probe captotate, and to detect the presence of the detector probe with optical techniques An ideal instrument to process the strips analyzers of Figure 1A is the VIDAS® instrument of bioMerieux Inc.

It will be appreciated that the choice of the instrument subsequent analytical to process the analyzer device It will depend on the design and the form factor of the analyzer device. The principles of the present invention of the amplifier station they are applicable to other form factors and, thus, this invention is not limited to any particular type of analyzer device or analytical instrument.

The detailed description of the design of the amplifier stations 200, in instrument 1 of figure 1, it will be understood more easily if the reader is already familiar with the design of the analysis device used by such stations, and with the theory of their operation. By consequently, in the next section of this document it is exposed a detailed description of the disposable analyzer device of Figure 1A processing the instrument 1. The characteristics Operational instruments 1 are fully exposed in sections Subsequent to this document and in the drawings that begin with Figure 18. It should also be noted that both parks amplifiers 200, located behind the two boxes 3 of the Figure 1A, are identical, and therefore, in this document only describe one of the amplifying stations. To the degree in that the two amplifier stations share common components of an electrical or compressed air system, those characteristics They will also be explained.

II. Detailed explanation of the construction and operation of the disposable analyzer device

With reference to figures 1, 1A, 2 and 3, the amplifier station of figure 1 is designed to receive a test strip 10 with a reaction vessel 12 of two cameras The reaction vessel 12 has a single dose, or unit dose of reagents for a reaction that is typical require different heat and content settings, such as is the nucleic acid amplification reaction (for example, the TMA reaction), packaged ready for use. The vessel of the Two-chamber reaction is designed as a disposable unit, single use only. It is preferable that the reaction vessel is completely molded into an analyzer device, such as a strip 10, with a set of wells 13 of wash reagent and detection, for use in a stand-alone detection station product of the amplification reaction (hybridization). By way of alternately, the reaction vessel 12 can be manufactured the same as an independent unit with flange or other convenient structures so that they can be installed in a designated space provided in such analyzer device

Two separate chambers, A and B, have been arranged in the double chamber container 12. The two main reagents, inside the reaction vessel, they have been stored in a way spatially separated. A chamber, chamber A, has the reagent sample / thermostable amplification (containing tabs, nucleotides and other necessary salts and compensating components), and the other camera, camera B; contains enzymatic reagents thermolabile, for example T7 and RT. Alternatively, the thermolabile enzyme reagents can be stored in a chamber intermediate or intermediate well in fluid communication with the second chamber, such that a reaction solution, coming from the first chamber, flow through the intermediate chamber en route to the second camera

The two cameras are linked by means of a fluid channel or connection duct 50 extending from the First camera to the second camera. A means is planned for control or allow the passage of fluid through the fluid channel from the first camera to the second camera. They have been contemplated various means for fluid flow control, such as the placing a valve in the fluid channel, as described in U.S. Pat. 5,786,182. In it they are described Several different embodiments of the valve.

A technician loads a sample of fluid into the first camera A and installs the test strip 10 inside the box 3 of instrument 1 of figure 1. A thermoelectric system Temperature control heats, inside the station amplifier 200, the first chamber only up to a temperature of denaturation (for example, 95 degrees C). The first camera, after the amplifier reagents in the first chamber have reacted with the fluid sample and the process of denaturation is complete, the first chamber is cooled quickly up to 42 degrees C for annealing the presser. The two chambers of the reaction vessel are not in fluid communication with each other before the stage of denaturation and cooling. After these stages are have completed, the means to control the passage is started of the fluid in order to allow the reaction solution go through the first channel 50 from the first camera A to the second chamber B. For example, the valve in the fluid channel is opens and the fluid sample is directed into the second chamber by pressure or vacuum techniques. So, the dissolution of the reaction is brought into contact with the enzyme (s) amplifier (as) (for example, T7 and / or RT) and the procedure Enzyme amplification continues in the second chamber B to 42 degrees C.

In a preferred embodiment, after the termination of the amplification reaction in chamber B, the analyzer device is removed by hand from the amplifier station 1 of Figure 1 and is inserted into a separate type instrument detection. In this detection type instrument a SPR® device (a fluid transfer device that serves as a solid phase receptacle), similar to a pipette, in the second camera The test strip 10 contains a plurality of wells arranged in a row. Hybridization, washing, optical analysis and decontamination continue then in the wells 13 according to well known techniques in order to detect the amplification products. Such processes may occur in the adjacent wells of a test strip of the containers of the reaction with two cameras automatically on the VIDAS® instrument bioMérieux, Inc.

Returning now to a detailed description of the construction of the analyzer device used in the station amplifier, Figure 1A is a perspective view of the analyzer device in the form of a strip 10 incorporating a reaction vessel 12, with two chambers, for the reaction of nucleic acid amplification that meets the requirements previous. The test strip 10 includes a plurality of wells of hybridization and washing 13, and an associated cover member 14. It is it is preferable that the test strip 10 of Figure 1 be made with a molded polymeric material, such as polypropylene.

A sealing membrane, such as a film of aluminum coated with polypropylene, applied to the surface upper 15 of the test strip to cover wells 13 and the reaction vessel 12, with two chambers, after the wells and container 12 have been preloaded with the enzyme, the reagent, washing solution or compensator, etc., appropriate. The membrane is not shown in figure 1A in order to better illustrate the structure of the test strip 10. The cover member 14 is sample before its annexation to the test strip in the vicinity of the reaction vessel 12, with two chambers.

The test strip of Figure 1A can be used in the amplifier station of figure 1 to perform a isothermal nucleic acid amplification reaction, by example, a TMA reaction, according to a possible embodiment of this invention. The chamber A of the reaction vessel 12, two chambers, contains the reagents or the amplification mixture, to know deoxynucleotides, tabs; MgCl2 and other salts and compensating components in liquid or granulated form. Chamber B is in fluid communication with well 52, of enzyme granules, which contains the amplification enzyme (s) that catalyzes (n) the amplification reaction, for example T7 and / or RT, in liquid or granulated form. In an alternative embodiment the amplifying enzyme is loaded directly into the chamber B.

After the addition of the objectives (or test tube) inside chamber A, the cover member 14 is closed on the test strip 10 in the manner to be described and the test strip is installed inside one of the boxes 3 of the instrument 1 of figure 1. Within the instrument, it is applied heat to chamber A to denature acid targets nucleicide of DNA and / or remove the secondary structure of the ARB. The chamber temperature A then cools rapidly for allow annealing of the presser. Then the dissolution of chamber A is brought into contact with the enzyme granules in the well for the granules 52 and the solution is introduced into the camera B. Cameras A and B, now in fluid communication with each other, they are kept at the optimum temperature for the reaction of amplification, for example, 42 degrees C. Spatially separating the chamber A of chamber B, and applying heat to the denaturation only to chamber A, thermolabile enzymes, in the well for enzymes 52, are protected against inactivation during the denaturation stage.

After the nucleic acid amplification reaction has been completed, the test strip 10 is removed from the instrument 1 of Figure 1 and processed within a second sensing machine adapted to process the test strips, such as the instrument VIDAS®. The test strip 10 of Figure 1A is given a particular shape factor (eg, contour, length, width, height, final configuration 18A and 18B, etc.) in order to enable the strip to be compatible. with an existing instrument base with a solid phase receptacle and other equipment to process the results of the nucleic acid amplification reaction in the test strip per se . In addition, the form factor of the test strip will stimulate the design of the mechanical characteristics in the amplifier station 200 of Figure 1. Thus, although the preferred embodiment of the test strip 10 has a suitable form factor for the base of the instrument of the assignee of the inventors, it will be appreciated that different sizes, shapes, configurations and other physical characteristics of the analyzer device incorporating the reaction vessel with two chambers can be reached, so that it adapts to other analytical instruments, and other instruments which would carry out the amplification reaction of the nucleic acid in the reaction vessel with two chambers. Therefore, the inventors do not consider this invention to be limited to the particular test strip illustrated in the drawings.

Figures 2 and 3 are additional views in perspective of the test strip 10 and the cover member 14 of the figure 1A. Figure 4 is an isolated perspective view of the cover member With reference to Figures 2 to 4, the member of lid 14 has a pair of elastic legs 20 with a configuration of wedge 21 that slammed over the protrusions 72A formed in the upper edge of the test strip, as will be explained more late along with figures 7 to 10. The legs 20 allow the back portion 22 of the cover 14 is firmly attached and security to test strip 10, while allowing a second front portion 24 of the cover 14 that is raised or lowered in relation to the back portion 22. The cover 14, manufactured with molded polymeric material, includes a hinge portion integral 26 that joins portions 22 and 24 together. This cover also includes a central opening 28 with a mesh filter porous placed in it to let the air into, or be remove from chamber A (after membrane removal sealer from the top of chamber A), while almost block the escape of the fluids or reagents from chamber A or the entry of foreign materials into chamber A.

The purpose of the cover 14 is for the user control access to chamber A and to provide a barrier of protection against the environment during the performance of the nucleic acid amplification reaction. During manufacturing of the test strip, the reagents are loaded into the chambers A and B (and in wells 13) and then a membrane is applied sealer to surface 15 of test strip 10, covering all wells 13 and chambers A and B. The membrane can be given a perforation or tear line at a location indicated in 34, adjacent to chamber A. Then the cover member 14 is installed on top of test strip 10. When the technician is ready to use test strip 10 the user lifts the front portion 24 of the lid to the position shown in figure 2. The edge 30 has a curved cavity configuration so that the finger of the user help lift portion 24. Then, the technician grabs the free edge 32 of the membrane (shown detached in the Figure 2 to illustrate the structure of the test strip) pushes the membrane out such that the membrane separates from the perforation indicated in 34. This action exposes the chamber A of the reaction vessel 12, with two chambers. So the technician inserts the sample of the fluid into chamber A and closes the cover member 14.

With reference to Figures 4A and 4B, with optimism and in the first embodiment, the film or membrane follows in place above chamber A. The cover member includes a button 41, manually operated, having a point or surface projection 41B on the lower side thereof such that when the user closes the cover member 14, can operate and tighten the button 41 and thereby make point 41B pierce the membrane which covers the top of the test strip above the chamber A to obtain a small opening for the introduction of the Sample of the analysis. In this embodiment the technician does not remove the thin membrane but rather is left in place. The action of button and the protrusion point is the mechanism by which You have access to camera A, at the time of use. This embodiment reduces the possibility that any fluid or reaction solutions intentionally migrate out of the chamber B and move into the environment. As seen in Figure 4A, the button 41 is connected to the rest of the lid by means of the legs elastic 41A which allow button 41 and the dot projection 41B move relative to the cover member and, of this mode, pierce the membrane. The side wall 41D of the button, once which has moved to the lower position, fits without strike within the corresponding circular wall portion 41E of the cover member 14, as best shown in Figure 4B.

Cap member 14 has one more pair of legs elastic grip 38 on opposite sides of it that is quickly hook onto tabs 72B at opposite edges of the test strip, resulting in the firm engagement of the lid 14 with the test strip 10. The legs 38 grip the strip 10 with much less force than the rear legs 20, so the cover 14 does not becomes completely disengaged from the test strip when the user lifts the front portion 24 of the lid. In the lid has been placed a third pair of legs 36 to contribute to align the front portion with the test strip 10 when the lid is closed.

With reference to figures 5 and 6, the surface upper 15 of the test strip 10 includes an opening 70 designed to accommodate a fork (shown in the figures 14 to 16) during the opening operation of the connection duct 50. The cover member 14 of Figures 3 and 4 is installed above of the test strip 10 such that the opening 40 of the member of cover is directly above the opening 70 of the strip of analysis. Also shown in Figure 5 are tabs 72A and 72B which allow the elastic legs 20 and 38 of the cover member 14 they close over the test strip when the cover member 14 is Install above the test strip. With reference to Figures 9 and 10, the test strip has an inclined portion 74 above which the wedge shape 21 (figure 4) of the member of lid slides until wedge 21 snags sharply under tab 76 and compressed against wall portion 78. The elastic nature of the legs 20 of the cover member and the wedge action 21 in contact with tab 76 prevents the cover member 14 get rid of the test strip during the lifting and lowering of the portion front 24 of the cover member. The inclined surface 80 of the Figure 9 helps to install the cover member and align the legs 20 in relation to the form of tab 72. The operation of the flange shape 72B is the same as that of the legs 38 of the lid 14.

With reference to figures 6 and 8, the strip of analysis has a pair of highlights 84 that extend in direction transverse and are molded at the bottom of the test strip to allow the test strip to be placed in an attitude stable, level, on the top surface of a table.

Figures 2 and 8 illustrate a hood of base 86 that is manufactured separately. Cap 86 is welded by ultrasonic means to the base of chamber A, to a frame 87 linking chamber A to connection duct 50, and to the base of the connecting duct 50. Cap 86 covers the most extreme portion bottom of chamber A and provides a fluid path for that the solution passes from the base of chamber A to the base of the connection duct 50, arranged vertically. The hood 86 is basically the same construction as the hoods that perform a similar function in the patent of the USA 5,786,182, which is incorporated herein by reference.

As best shown in Figures 5, 8 and 11, the test strip 10 also includes a pair of desiccant wells 54 and 56, which are placed in air or fluid communication with the chamber B. The desiccant well 54 is also shown in Figure 6, the what is a sectional view of the test strip taken along from line 6-6 in figure 5. Desiccant wells 54 and 56 are intended to hold one or a plurality of granules desiccants stacked on top of each other within their respective wells During the assembly of the test strip, the inspection Mechanics of desiccant wells will confirm the amount of granules desiccants in wells 54 and 56. The purpose of the desiccant is prolong the duration of the amplifying enzyme loaded on the strip of analysis, particularly when the amplifying enzyme has the granule form and is susceptible to degradation in the presence of a humid environment In the case that the nucleotides, the MgCl2, the sizing agents and other reagents, loaded in the chamber A, have a liquid form it is not necessary, then, that wells 54 and 56 are placed in direct aerial or fluid communication with chamber A. However, in case the reagents in the chamber A have a granulated shape or that are otherwise susceptible to degradation in a humid environment, then the desiccant wells will be designed and manufactured for communication with camera A in addition to camera B. Alternatively, it you can install a second set of desiccant wells adjacent to the chamber A to serve the reagents in chamber A.

With particular reference to Figures 6 and 11, the extreme lateral portion of the desiccant well 54 includes a passageway indicated with the number 58 that allows air communication with the chamber B (and the final air communication with the enzyme granule placed in the well of enzymatic granules 52). The passage 58 is installed on top of a wall 60 that separates the lateral portion of the desiccant well 54, from chamber B. In desiccant well 54, place three or four desiccant balls. Alternatively, the desiccant balls could be placed directly in chamber B (and in chamber A as needed), or mold inside the material form the reaction vessel 12 with two chambers.

After denaturation and annealing of the fluid sample presser have taken place in the reaction vessel A, at the first temperature of the reaction, a ball valve opens, indicated generally with the reference number 102 in figures 12 and 13. This valve It consists of a metal ball 104 that is arranged inside the duct of connection 50 in an intermediate region 106, cylindrical. The ball 104 has a size such that its diameter is equal to the diameter of intermediate region 106 and, of this, it is normal for it to form a complete obstruction of the connection duct. The walls 18 of connection conduit are made of a deformable material (and Propylene is, for the present purposes, enough deformable). The deformability of the walls 108 is such that, when the walls 108 are compressed on opposite sides of the ball 104, the wall 108 deforms perpendicular to the force of compression, on opposite sides of the ball, for, in this way, create a passage for the fluid around the ball.

In amplifier station 200 it has been installed a fork to create this deformation action on the walls 109 and ball 104 (as best shown in Figure 14). The fork 110 has two teeth112 for each position, that is, six forks with a total of twelve teeth per box, for one box intended to contain six test strips at any time. The fork 110 is lowered by means of the opening 40 of the lid (as best shown in Figure 14), through opening 70 at the top of the test strip (best shown in the Figure 11), such that teeth 112 come into contact with tighten with the walls 108 of the connection duct 50, directly on opposite sides of the ball 104, as shown better in figure 16. This compression action deforms the walls 108, as shown in Figure 17, to form corridors 116 in opposite sides of the ball 104.

Simultaneous with, or immediately after, the ball valve opening as just described, it extracts vacuum in the test strip, and in particular in the first reaction chamber A. This is achieved by placing an enclosure of empty around the test strip in boxes 3 of the station amplifier 1 (described in more detail below), and evacuating the air in the vacuum enclosure. Vacuum extraction lowers the pressure in both first chamber A and second chamber B, since who are now in fluid communication with each other. When releases vacuum there is a pressure gradient between chamber A and the chamber B, with chamber A at a higher pressure. The gradient of pressure forces the fluid solution to enter chamber A at through the passage in the hood 86 (see figures 12 and 13), up and around passages 116 in the conduit of connection 50, as indicated by the arrows in the figure 17, and up to the top of the connection conduit fifty.

Once the fluid solution has reached the upper part of the connection duct 50, the fluid enters a channel 100 (see figures 11 and 12) that leads to the well of the Enzymatic granules 52. The fluid dissolves the enzymatic granules  130 (figure 12) in well 52 and carries the amplifying enzyme inside chamber B. The amplification of the nucleic acid in the fluid sample occurs in chamber B at temperature specified, for example, 42 degrees C.

With reference now to figures 14 and 15, the alternative movement action of the fork 110 opening the Ball valve, shown schematically. In figure 14, the test strip 10 is shown with the sealing membrane 42 applied to the top surface of test strip the way described above, as it should be when the device is manufacture and be ready for use. The membrane 42 carries a code of bars 43 that identifies the type of test strip that is being using or other pertinent information.

Figure 15 shows the characteristics basic operation of the forks 110 at the station amplifier 1 of figure 1. Figure 15 shows two test strips installed in the amplifier station, which shows in a front view and partly in section. The Forks 110 are shown to be integral with the crossbar 132 the which, in turn, is bolted to the top of a wrap of vacuum cover 134 at the amplifier station 1. The strips of Analysis 10 are installed in a set of TEC and heat sink 136 that holds the two chambers of the reaction vessel, with two chambers, in the test strips at the appropriate temperature, as described in detail in US Pat. 5,786,182. The vacuum cover casing 134 is attached to a mechanism of mechanical transmission that raises and lowers the cover of the vacuum lid in relation to a lower support structure 138. The shell of the cover 134 and the support structure 138 define an enclosure or vacuum chamber 140. The vacuum lid casing 134 includes also ports (not shown) to extract air from the enclosure of vacuum 140 and reintroduce air into the vacuum enclosure 140. When the vacuum cover casing 134 is lowered over the support structure 138 forms an air tight closure with the support structure 138 (using a seal structure appropriate in region 139), allowing the vacuum to be extracted in The enclosure. The removal of vacuum in room 140 causes it to Extract air from the reaction vessel with two chambers through of the opening 28 in the cover member 14 and a filter permeable to air 142 placed inside it (see figure 14). Then, when the vacuum is released in room 140 (remaining the wrapped 134 in the lower position during the release of the vacuum) the pressure difference between chambers A and B causes the fluid solution in chamber A migrates through the conduit of connection, open by the action of the forks 110, and enter the chamber of enzymatic granules and in chamber B, as before described.

Additional details of the test strip 10 now preferred are set forth in US Pat. 5,786,182.

II. Detailed description of the station amplifier Compendium

With reference now to figures 18 and 19, the top cover of amplifier module 2 is shown ginned with the in order to better illustrate the two amplifier stations 200, identical, placed immediately behind the boxes 3. The module amplifier 2 also includes a pair of glass bells 202 and the corresponding ones associated with a compressed air system 204 for stations 200, which are described below with reference to figure 46.

One of the amplifying stations 200 of the Figures 18 and 19 are shown in a perspective view in the figure 20. Figures 20 to 27 are a set of views in elevation, in plan and in perspective of amplifier station 200. With reference to these figures, together with figures 1 and 2, the amplifier station includes a support structure 206 that is adapted to receive from one to six disposable analyzer devices 10 of the Figures 1A to 17. In particular, the support structure 206 includes a set of raised erect elements 208, each of which has a groove 210 (figure 21). Grooves 210 extend the length of the protrusions 208 and receive the shapes cylindrical projecting outward at end 1B of the analysis strips 10 of figure 2. The analysis strips are enter by hand in box 3, entering end 1B first, so that the strips are held in place by the action that ends 1B are held by means of grooves 210 within the raised shoulder elements 208.

The amplifier station 200 includes a system of temperature control for the test strips. This temperature control system is described with reference to Figures 34, 35, 37 and 38. Basically the control system of the temperature consists of thermoelectric heating elements, the associated heat sinks and a control system feedback. The temperature control system maintains the chamber A of the test strip at a high first temperature in order to denature the sample in chamber A of the test strip 10. The temperature control system simultaneously maintains the amplifying enzyme in the well of enzymatic granules at a second temperature lower than the first temperature, in order to preserve the second reagent of nucleic acid amplification (i.e. avoid inactivation of the amplifying enzyme). The temperature control system also keeps chamber B of the test strip at temperature that is desired for the amplification reaction performed within the same. The thermoelectric heating elements are placed in thermal and electrical contact with support structure 206 immediately adjacent to the test strips and transfer heat to, or remove heat from, the reaction chambers of the analysis strips

The amplifier station 200 also includes a actuator that is operative in test strip 10 to place the first and second chambers of the reaction in fluid contact between yes. The actuator is operational in the test strip after there has been a reaction in the first reaction chamber A a The first high temperature. The constructive form of the actuator It will vary depending on the design of the analyzer device. In the preferred embodiment of the test strip, the actuator consists on a fork 110 with two teeth or spikes 112. In the embodiment present there are six such forks 110 (one for each strip of analysis). The forks are best shown in figures 24 and 28. These forks are mounted on the top surface of a vacuum wrapped 134, and they have alternative movement up and below with the vacuum envelope 134 in relation to the structure of support 206 and the test strips in the manner described below with more detail.

The amplifier station includes, in a preferred embodiment, a compressed air system that activates the transfer of a reaction solution from the first chamber A of the test strip to the second chamber B. An implementation possible of the compressed air system is to use the vacuum probes that draw a vacuum in the second chamber B of the test strip. The vacuum draws fluid from chamber A, through the conduit of connection 50, in the test strip, even within the second chamber B. This technique is described extensively in the patent of The USA. 5,786,182, which is incorporated herein by reference.

The entire test strip and, indeed, all of the six test strips are placed, in a most preferred embodiment, within a vacuum enclosure and extracted empty in the test strip. The vacuum release causes the fluid is transferred from chamber A to chamber B due to the pressure difference between the two chambers. The station amplifier 200 includes a compressed air system (illustrated in figure 46 and described below) that generates the release and releases a vacuum in a vacuum enclosure defined by the envelope of the upper vacuum chamber 134 (see figures 23 to 26) and the support structure 206. The vacuum chamber envelope upper 134 rises and falls by means of a transmission system between an elevated position, shown in figure 23, and a position more low. In the lowest position, a seal 220 (Figure 29C) held in the retainer 222 of the seal Hermetic vacuum chamber casing 134 sits on a peripheral planar surface 224 of the support structure 206. The seal 220 forms a seal while in the air between the envelope of the vacuum chamber 134 and a structure of support, allowing a vacuum to be removed inside the shell of the vacuum chamber. When the vacuum chamber envelope 134 it is lowered onto the surface of the support structure 224, the forks 110 act to open the valves of the strip analysis in the manner indicated in figure 15. As this is evident from figures 20 to 27, all the test strips, loaded into the amplifier station are subjected, with simultaneity, to the action of the valve and to the transfer Pneumatic fluid from reaction chamber A to chamber of reaction B in the test strips.

It is important that support structure 206 and, in particular, the peripheral surface 224 thereof, are completely leveled, so that when the vacuum wrapped 134 on the support structure 206 the gasket of sealing 220 form a tight seal. It has been discovered that, loosening a collar 226, at the top of the screws guide of the vacuum sheath drive system, the vacuum wrapped 134 has enough slack for a uniform way it is held in the support structure and form a vacuum shutter.

Additional mechanical characteristics of the station 200 amplifier

The amplifier station, with reference to the Figures 20, 22 and 23, include a door 230 which, by means mechanics, is attached to the vacuum casing 134 and has movement alternative above and below it. When the door goes up up to the position shown in the drawings the user can insert the test strips into boxes 3 of figure 1 and within projections 208 of support structure 206. Also there is a sensor plate 232, mechanically attached to the housing of emptiness. The sensor plate 232 has a flange 234 that rises and falls within an opening 236 in a structural support member 237. Three optical interrupt sensors 238A, 238B and 238C are mounted on a side panel 240 of the station and detect the passage of the upper and lower edges 242 and 244, respectively, of the sensor plate 232. Optical interrupt sensors 238 A to C carry signals to the electronic control system for the station and they are used to monitor and control the rise and fall of the door 230 and vacuum housing 134.

The top of the amplifier station includes a 250 tray with an optional 252 air filter. The air filter 252 filters the air in the air inlet pipe 254 leading to vacuum casing 134. Tray 250 carries also two solenoid valves 256A and 256B that control the vacuum extraction and release in pipes 254 and 258 that lead to vacuum casing 134. The operation of the 256A and 256B valves will be analyzed later. A pipe 260 goes from the port 292 of the vacuum chamber envelope, illustrated in figures 26 and 27A, up to a pressure sensor that monitors the pressure inside the vacuum chamber envelope.

With reference now to figures 21, 24 to 26 and 31, on top of the support structure 206 a set is mounted of optical reader 207. The optical reader assembly 270 includes up to six optical sensors per position that are positioned directly above spaces 272 between projections 208 in the structure of support 206. Optical sensors detect if the user has introduced an analysis strip into the support structure 206, since such strips will occupy spaces 272. The set of optical reader is shown isolated in several views in figures 32A to 32D.

With reference to those figures but mainly to Figure 31, the optical reader assembly 270 includes a cable 274 for optical sensors. Cable 274 has a male plug 278 that connects to another cable that leads to the control system electronic for the station. Cable 274 leads to a housing 276 that is received in an opening in the support structure 206. The housing 276 is retained within the support structure 206 by means of a C-shaped clamp 280. A gasket of tightness 282 prevents air from escaping around the side of the 276 housing during vacuum operations. 274 cable leads to six sets of 284 optical sensors located within a cover 286.

With reference to figures 26, 27 and 31, the vacuum wrapped 134 includes a protruding portion 290 that receives the housing 276 when the vacuum envelope is lowered above the support structure 206. Vacuum casing 134 is shown isolated in figures 29A and 29B. Vacuum casing 134 includes three ports 292, 294 and 296. The port 292 receives a tube 250 (Figure 20) leading to a pressure sensor that monitors the air pressure inside the vacuum casing 134 when the envelope 134 is lowered on top of support structure 206. The port 294 receives the tube 258 that leads to the valve solenoid 256B of Figure 20. The air is drawn from the enclosure of vacuum provided by the vacuum envelope 134 across the louver 294 and its associated tube 254. Louver 296 receives a third tube 258 leading to solenoid valve 256B of the Figure 20. The air is reintroduced into the vacuum enclosure via the port 296.

The vacuum envelope 134 also receives a sensor of temperature 300, of negative temperature coefficient. In this type of sensor, when the temperature rises, the value of the resistance decreases. The temperature sensor 300 has the conductive wires 302 that conduct voltage signals to the system electronics and temperature information for the station, described in more detail below. Basically the information on results provided by the ambient temperature sensor allows compensation of a deviation in the temperature of the structure of support due to the heating of the ambient air inside the vacuum chamber.

With reference to figures 20, 23, 28, 29A and 29C, vacuum casing 134 also includes openings 304 for receive a pair of bolts 306. Bolts 306 hold the envelope of vacuum 134, cross member 132 and forks 110 in a member of 308 support oriented horizontally. A couple of joints O-rings 309 prevents air from entering around crossbar 132 in the vicinity of bolts 306. Support member 308 is attached on opposite sides of it to a guide collar 310 that goes up and down by the rotating action of a 312 regulating screw driven by a motor and belt drive system that, in general, is indicates with the number 314. See also figure 40.

With reference now to figures 20, 21 and 23, there are a couple of 320 fans installed in the lower portion of the station. Fans 320 direct air into the space below of the horizontal support member 206, and, in particular, above a set of fins 322 that provide a heat sink for thermoelectric elements in the temperature control system for the season

With reference now to Figures 36, 33A and 33B, the entire support structure 206, including the fins Heatsinks attached 322, shown insulated in views in perspective. Figure 33C is a plan view from the top of support structure 206. Support structure 206 includes a support tray 207. This support tray 207 includes three 324 guide collars, two on one side and one on the other. The collars guide 324 receive a shaft that extends from the front of the station to the back of the station. The axes are shown in figures 24 and 25 with reference number 326. As shown in Figure 36, guides 324 include a part Insert 328, low friction plastic. A coil spring 330, which is best shown in Figures 20, 22 and 23 is arranged between the end of the guide collar and the superstructure of the station. The coil springs 330, the guide collars 324 and axes 326 allow the entire support structure to move back and forth along the axis of axes 326 for the stirring and mixing operations so that the dissolution of the reaction in the test strips completely dissolve the enzyme granule. The action of the alternative movement of the support structure 206, for stirring operations and mixed is, provided by a set of engine, belt and gear eccentric, described in greater detail below.

The support structure includes, according to illustrated in figures 33 B and 33C, a pair of vertical tabs 340. Cover 286 of the optical reading assembly 270 of the figure 32A, is mechanically attached to eyelashes 340. Of this mode, the sensors of the optical reading set are positioned directly above the 272 spaces between the raised projections 208. The six pairs of regions are also illustrated in Figure 33C recessed 342 in the front portion of the support structure4 206. The recessed regions 342 are intended to allow the teeth 112 of the forks 110 (figure 28) are inserted by complete within the test strips, without perching on the basis of the support structure 206 and damage the forks. In the figure 33C also shows an opening 344 in the support structure receiving housing 276 of the optical reading assembly (see Figure 31).

With reference now to figures 28 and 30A a 30D, cross member 132 and forks 110 are shown insulated. He crossbar 132 has a pair of cavities 354 to receive a joint O-ring 309 (figure 28) that forms a tight closure for the envelope of emptiness. A cylindrical erect figure 355 receives bolts 306 of Figure 28 that tie the crossbar to the main member of horizontal separation The crossbar 132 and the integrated forks 110 and teeth 112 are manufactured with good stainless steel quality in order to withstand the forces that are required to open six of the ball valves in six test strips, during as long as the instrument lasts.

The crossbar 132 also includes a set of six 360 positioner teeth, spring actuated. Teeth 360 positioners can be moved inside a cylindrical cavity 362 on crossbar 112 against the force of a spring deflection 364. Positioning teeth 360 press down the cover 14 of the test strips 10 (figure 2) to help the cover 14 to form a seal around chamber A in The test strip. The purpose is such that when air is evacuated of cameras A and B of the test strip, during the procedure of the vacuum, and then reintroduce into the chambers when release the vacuum, air pass through the porous mesh filter 142 (figure 14) on the lid 14 and not around the edges of the lid. The springs 364 limit the amount of force applied to the cover 14 at approximately 1,362 kilos when the fork and chamber of vacuum are lowered over the analysis strips and the structure of support 206, preventing the lid from breaking.

With reference now to figures 24 and 25, the station 200 sits upright inside amplifier module 2 of Figure 1 by means of two legs 352 and a pedestal 354.

Functional characteristics of the control system of the temperature

With reference to Figures 33B, 34 and 35, will now describe the general operation of the control system of temperature. Figure 34 is a bottom-up plan view. of station 202, with all drive motors and others disassembled components in order to show more clearly the basic characteristics of the temperature control system. The support structure can be 206, in its concept, divided into two temperature controlled regions, a first region 370 and a second region 372. Region 370 is dedicated to the warming of the A chamber of the test strip, until a first temperature high, for example, ≥ 65 degrees C, for denaturation of the sample. Region 372 is dedicated to the warming of the chamber B of the test strip, up to a second temperature lower than the first temperature, in order to preserve the integrity of the amplifying enzyme in the granule well enzymatic and perform an amplification reaction in chamber B of the test strip at the desired temperature, for example, approximately 42 degrees C.

Region 370 is maintained at a first temperature under two cooling elements thermoelectric (TEC) 374A and 374B that are in physical contact and thermal with the front of the projections that support the strips of analysis. The 374A and 374B thermoelectric refrigerators are in physical and thermal contact with heat dissipating fins 322. The 374A and 374B thermoelectric refrigerators are positioned between fins 322 and the upper surface of the structure of support, as will be described later with reference to Figures 37 and 38. Some thermosensitive resistors, that is, thermostats, embedded in the support structure and in the heatsinks provide results information to the computer control system

Similarly, the temperature of region 372 is controls by means of two 376A thermoelectric refrigerators and 376B, in physical and thermal contact with the rear portion of the Highlights that hold the test strips and fins chillers or heat sink 322.

Figure 35 illustrates, in schematic, the operation of thermoelectric refrigerators. By way of basic, a thermoelectric refrigerator is a device in state solid that works just like a heat pump without any part mobile, fluids or gases. The thermoelectric coolers are formed by two semiconductor elements, mainly telluride of bismuth, with many impurities to create an excess (type N) or a deficiency (type P) of electrons. The heat absorbed in the joint cold is pumped to the hot joint at a rate proportional to the current passing through the circuit and the number of thermocouples. The electrons, in the cold junction, absorb energy (heat) as passing from a low level of energy in semiconductor element type P, towards a high level of energy in the semiconductor element type N. The power supply of c.c. supply the energy to move electrons throughout the system. The energy is ejects a custom heat sink in the hot joint that electrons move from the high level element of energy (type N) towards a low energy element (type P). By reversing the polarity of the source of DC power The heat sink becomes the source of Heat and heat source becomes the heat sink. From in this way the thermoelectric coolers of figures 34 and 35 they can be used both for heating and for cooling the structure Support and analysis strips according to the profile of desired temperatures for an amplification reaction of nucleic acid. 374A thermoelectric cooling elements, 374B, 376A and 376B of Figure 34 are available in the Commerce.

With reference to Figure 37, the structure of support 206 and the temperature control system are shown in a sectional view taken along the line 37-37 of Figure 34. Figure 37 shows two TEC 374A and 376A modules positioned immediately above, and in thermal contact with, the fins (heat sinks) 322. The front TEC module 374A has the responsibility of carrying the front portion of support structure 206, in region 370, up to a typical first temperature greater than 65 degrees, as indicated above. In the same way, the TEC 376A module is in thermal and physical contact with the rear set of dissipating fins of heat 322 and maintains region 372 of the support structure at a second temperature, for example, 42 degrees C.

Figure 38 is a more detailed sectional view. of regions 370 and 372 of Figure 37. A thermistor 400 is recessed in heat sink 322 and monitors the temperature of the heat sink for the control information system temperature. The TEC 374A module is sandwiched between the heatsink 322, an electrical insulator 401 and a tray plastic 402 forming the front portion of the support structure 206. A 404 bolt holds the assembly 322, 374A and 206. A gasket of tightness 406 prevents air or fluid from escaping around the plastic tray 402. A second thermistor 408, embedded in the frontal region 410 of the raised shoulder 208, monitors the support structure temperature in the region immediately adjacent to chamber A of the test strip. The second thermistor 408 is mounted inside the raised shoulder 208 by means of a plastic platform 412 extending from one side to the other of the support structure and is held in place by means of a fixing set 414.

The 412 platform and a second set of 416 fixing also hold a third 418 thermistor. The two Thermal regions 370 and 372 of the raised shoulder 208 of the structure support are separated from each other by means of a separator Insulation Delrin 420, air spaces 422 and screw 424 locator.

With reference to the left side of Figure 38, the rear thermal region 372 includes the TEC 376 A, and an insulator electric 426, an o-ring seal 428 and a clamp 430 that holds the whole set.

With reference again to Figure 37, you will see that the raised shoulder 208 of the support structure 206 includes a 432 aluminum thermoconducting block for the thermal region rear or "amplifier" 372 (for camera B of the strip analysis and amplifying enzyme), and a second block 374 aluminum thermoconductor for the front thermal region or "of the sample" 370 (for camera A). The chosen material for the most posterior portion 436 of the raised shoulder is not, in particular, important since it does not perform any function of heat transfer in the illustrated embodiment.

Figure 37 also shows a card 433 printed circuit containing the electronics for both Sample 320 fans in Figure 34 and modules 376A, 376B and 374B and 374B.

With reference to figure 39, the support structure 206 and the components associated with the system temperature control, in another sectional view taken along of line 39-39 of Figures 33C and 34. The Full heat sink sample 322 is mounted on the thermal block of sample 434 by means of bolts 440 and 404. Tension spring 442 and a gasket have been installed tightness 444 on opposite sides of the assembly to limit the amount of force applied to TEC 374A and 374 B modules through of bolts 440 and 404.

Functional characteristics of the stirring system and belt drive

Figure 40 is a perspective view of the superstructure of station 200 with most pieces of the same disassembled in order to better illustrate the system of station transmission. Transmission systems consist of two independent sets: (1) a transmission system by 500 belt to raise and lower the vacuum chamber envelope in relationship with the support structure and (2) a system of 502 stirring drive to produce movement alternative of the support structure along the axis of the axes 326 (see figure 22).

With reference to figures 40, 28, 42 and 43, the 500 belt drive system includes a speed engine variable 504 that drives a toothed belt 506 that spins a 508 gear pair and corresponding regulating screws 312. The rotation of regulating screws 312, inside the collar 310, makes the horizontal support member 308, collar 310 and the attached vacuum chamber housing assembly 134 and fork 110 rise and fall in relation to the screws regulators The optical sensors 238A to C of Figure 22 detect the position of the transmission system 500 when monitoring if the detector panel 234 is obstructing the path of light from one side to other sensor.

With reference to figures 26, 40 and 42 to 44, the 502 agitation drive system includes a motor variable speed 550, a toothed belt 554 and a set of eccentric gear 556. An optical sensor 558 detects the position of a circuit breaker 560 inside a disk 562 attached to gear 556 and generates a signal that the 550 engine uses to make the gear Eccentric return to its starting position. Eccentric gear remains in contact with a block 564 mounted on the underside of support structure 206 (best illustrated in the figures 28, 33B and 37) and remains in contact with block 564 by the action of the coil springs 330 (figures 23 and 25) surrounding to shafts 326. The rotation of eccentric gear 556 causes the alternative movement of the entire support structure 206, causing the stirring movement to be imparted to the strips of analyzes loaded on the support structure, facilitating the complete dissolution of the granule and encouraging the mixing of the reagents with the fluid sample in the test strips.

The speed of the stirring system is, approximately 8 to 10 hertz, with a 3 mm stroke ± 1.5 mm. Agitation occurs for 60 seconds and begins when the drive system 500 raises the forks and the wrapped in the vacuum chamber, after the fluid has run transferred from chamber A to chamber B in the strips. From in this way the stirring promotes the reaction between the dissolution of the reaction from chamber A with the amplifying enzyme.

As illustrated in Figures 43 and 44, the eccentric gear 556 extends through an opening in the base or platform 570 for the station. The 570 base includes a pair of upright guides 572 to support the shafts 326 of the figure 22

Functional characteristics of the electronics system

With reference to Figure 45, the system of 600 electronics for the amplifier station is shown in shape of functional scheme. The electronics system 600 includes a front tray card 602 that receives signals from passive temperature sensors at the front of the support structure corresponding to temperature region 370 and supplies these signals to a tray interface card 604. A back tray card 606 receives the signals from passive temperature sensors in the portion rear of the support structure corresponding to the region of temperature 372 and supplies them to the interface card of the tray 604.

The 610 servo card controls the components station assets, including vacuum valves in the compressed air system, fans, and motors for transmission systems The 610 servo card also issues commands to the optical sensors in the optical reading system to detect if the strip has been loaded into any given slot of the support structure. There is a 610 servotecard per box.

A 612 interface card has the responsibility for a variety of tasks, including control of the servo card, via the RS 485 communications interface, the communication with computer system 5, for applications general, from figure 1, vacuum feeding, management Ready and Current LEDs On. Interface card 612 includes a 68HC11a microcontroller, a flash memory that stores computer system software every time the station turns on, a RAM that saves program data, a drive disk and receiver that provides the interconnection between the microcontroller and computer system 5, a reference of the tension, which provides a measurement of the vacuum inside the tanks Vacuum compressed air system, the MOS transistors that provide power for the vacuum pump and discharge valves to the atmosphere and a fuse that provides a 12 volt protection

The details of the electronics system are not consider relevant for the present invention and any person skilled in the art can develop them with ease.

The 610 servo card controls the entire temperature cycle procedure sent by the card interface 612. The four sensors inside the instrument (sensor the ambient temperature of the vacuum chamber, sensor of the heatsink temperature and temperature sensors the front and rear regions in the support structure) provide measurements to control the process of the temperature. All these sensors are coefficient thermistors negative temperature (NTC), as indicated above. The temperature acquisition is done by means of a microcontroller in a servo card that selectively calls a 12-bit analog to digital converter for the value of any of the temperature sensors. The tension value represents the impedance of the sensor, which can be correlated With a temperature reading.

The temperature control system includes plus four field effect transistors metal-oxide-semiconductors which provide to each TEC module positive or negative voltage. Microcontroller on the 610 servo card operates a memory code that controls the eight field effect transistors metal-oxide-conductors. Each TEC is controlled by independent way.

Functional characteristics of the pneumatic system

The pneumatic system 204 of Figures 18 and 19 is shown schematically in figure 46. System 204 gives service to both boxes in instrument 1. This system includes the vacuum wrapped 134 forming an enclosure around the strips of analysis 10 and support structure 206, a vacuum circuit 700 indicated with the continuous line in figure 46 and a circuit of atmospheric pressure 706 indicated with dashed lines.

The vacuum circuit 700 includes a pump 704 vacuum that maintains a vacuum (50 kPA) inside the two tanks of vacuum 202. A vacuum sensor 706 measures the pressure within the vacuum tanks This circuit also includes a stop valve to the EV3 atmosphere. Vacuum tanks 202 are attached to the vacuum wrapped 124, for the two boxes, by means of a reducer of current 708, a splice in T 710, and vacuum pipes leading to the EV1 vacuum valve (position 256B in Figure 20) and the vacuum tube 258. Each vacuum casing 134 has a tube 712 which leads to a vacuum pressure sensor 714 that monitors the vacuum inside the vacuum casing 134 when it is lowered over the support structure 206.

The vacuum circuit 700 works as follows: when the EV2 solenoid valve is closed the vacuum shell is At atmospheric pressure When the EV1 valve is open, the air inside the vacuum shell flows into the deposits of vacuum 202 through the reducer of the current 708. The reducer of current 708 guarantees a gradual decrease in pressure inside of the vacuum envelope 134.

Atmospheric pressure circuit 702 includes an EV2 airflow valve (position 256A in the figure 20) for each box, a tube 254 that leads from the vacuum shell 134 to a filter 252 and the valve EV2, and a reducer of the current 716.

The atmospheric pressure circuit works as follows: the EV1 solenoid valve is closed and the vacuum inside The vacuum envelope is 50 kPa. When EV2 solenoid valve is open the ambient air flows to the vacuum envelope to through the reducer of the current 716 and the filter 252. The reducer of current 716 guarantees a gradual increase in pressure inside the vacuum envelope 134.

During the initialization of station 200, the programming for the instrument opens the valve to the EV3 atmosphere to record the deviation of the vacuum sensors 706 and 714 at the current atmospheric pressure.

Thermal cycle

Chamber A of the test strips is heated or cools by means of the two TEC 274A and 274B modules before described. The same heat sink allows the dissipation of the heat from the TEC modules. Similarly, the two TEC 276A and 276B modules heat and cool chamber B of the amplification reaction of the test strips and the heatsink of heat and heat sink alerts, coupled to TEC modules 276 A and 276B allow heat dissipation from these thermoelectric refrigerators

Figure 47 shows the thermal cycle process that amplifier station 200 carries out for a representative embodiment of the nucleic acid amplification reaction for an amplified analysis of the Chalmydia trachomatis . At time t o the temperature of the front portion of the support structure rises to a denaturation and annealing temperature of the sizing of approximately 95 degrees C and is maintained there for approximately 10 minutes. At time t_ {1} the temperature drops rapidly from 95 degrees C to 42 degrees C. At time t_ {2} the reaction solution is transferred from chamber A to chamber B in strips of analysis (the vacuum chamber is lowered above the test strips and the vacuum procedure described above occurs). From time t 2 to time t 3 (approximately 60 minutes), an amplification reaction occurs in chamber B of the test strips. The temperature in chamber B rises rapidly at time t 3 to an inactivation temperature of 65 degrees C at time t 4 and remains there for 10 minutes until time t 5. At time t5 the temperature is reduced to an inactivity temperature of 37 degrees C until the procedure is repeated. Then the test strips are removed from the boxes 3 and inserted into another instrument to process the amplification products with a probe, solid phase receptacle or other equipment.

Alternative realizations

A possible alternative embodiment is to couple the support structure in the boxes to another drive system move the support structure in relation to the door of the box between a retracted position and an extended position. This Transmission system could be of any appropriate design. In the extended position the support structure is projected within the door opening or even beyond out, enabling, for the user, much easier access to the support structure and to install the analyzer devices in the support structure. When the user has loaded the analyzer devices these will indicate, in the interface of the user, that the support structure has been loaded after which support structure is removed by means of the system of transmission inside the box to the position shown in the Figures 20 and following.

The amplifying station may, for certain reactions, as in other examples, that only need to maintain a temperature region in the device analyzer, namely, to maintain the second reaction chamber at a reaction temperature such as 42 degrees C. Thus, in instead of two TEC devices and their associated heat sinks only a TEC device and the corresponding heat sink are installed in the amplifier station adjacent to chamber B in the strips of analysis.

Claims (10)

1. A station to carry out a reaction of nucleic acid amplification in an analyzer device disposable with a first reaction chamber containing a first nucleic acid amplification reagent and a second chamber of reaction containing, or in fluid communication with, a second nucleic acid amplification reagent, the station comprising: a support structure to receive said analyzer device; at least one temperature control system for said analyzer device, said control system of the temperature maintaining said first reaction chamber at a first temperature but simultaneously maintaining said second nucleic acid amplification reagent to a second temperature different from said first temperature in order to preserving said second amplification reagent of the reaction; an operating actuator in said device analyzer to place said first and said second cameras of reaction in fluid communication with each other, said actuator being operational on said analyzer device after a reaction has taken place in said first reaction chamber to said first temperature; whereby a second portion of said reaction nucleic acid amplification may occur in said second second chamber with said second acid amplification reagent nucleic being preserved by maintaining said second nucleic acid amplification reagent to said second temperature while said first chamber and said first nucleic acid amplification reagent are maintained at said first temperature, and a pneumatic system that works in said analyzer device for extracting a reaction solution from the first camera to the second camera after the actuator said device has acted to place said first and second cameras in fluid communication with each other, said system comprising pneumatic a vacuum enclosure and a vacuum source, said vacuum enclosure comprising a wrap movable with respect to said support structure and said analyzer devices to thereby enclose said analyzer devices and allow a vacuum to be removed in said analyzer devices; said vacuum source placed in communication with said vacuum enclosure and running to extract air from said enclosure and admit that air enters said enclosure and thus transferring said reaction solution from said first chamber to said second chamber. 2. The station of claim 1, wherein said analyzer device comprises an elongated strip having (1) a reaction vessel comprising said first and second chamber and a connection conduit that links said chambers, and (2) a plurality of wells. 3. The station of claim 1, wherein said support structure houses a plurality of said analyzer devices and in which said control system of the temperature and actuator are operative in each of said plurality of analyzer devices, whereby said instrument is capable of processing said plurality of devices analyzers substantially simultaneously. 4. The station of claim 1, wherein said station further comprises a mechanical stirring subsystem coupled to said support structure by agitating said devices analyzers after said analyzer devices are have installed in said support structure. 5. The station of claim 1, wherein said station further comprises a front panel having a door providing access to said support structure, and in which said support structure is coupled to a system of transmission to move said structure in relation to said door between a retracted position and an extended position, allowing said extended position that a user comfortably installs said analyzer devices in said support structure. 6. The station of claim 1, wherein said temperature control system comprises: a first thermoelectric element installed in said instrument in the vicinity of a first portion of said support structure, wherein said first element thermoelectric keeps said first chamber to a first temperature, and a second thermoelectric element installed in said instrument in the vicinity of a second portion of said support structure, wherein said first element thermoelectric keeps said second chamber to a second temperature. 7. The station of claim 6, further comprising first and second heat sinks in thermal contact with said first and second elements thermoelectric, respectively. 8. The station of claim 7, wherein said support structure comprises a pair of cavity shapes elongated receiving a form of projection on said device analyzer to thereby allow a technician to slide said analyzer device towards said support structure and, of this mode, precisely position said analyzer device in relationship with said first and second thermoelectric elements. 9. The station of claim 1, wherein said actuator comprises a plurality of forks adapted for engage a plurality of said analyzer devices installed in said station and open a connection conduit linking said first and second reaction chambers. 10. The station of claim 9, wherein said plurality of forks are fixed with respect to said vacuum enclosure and have alternative movement with said enclosure of vacuum between open and closed positions in relation to said support structure.